Spontaneous, rhythmic subsarcolemmal local Ca2+ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca2+ releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca2+ and cAMP/PKA regulatory components of automaticity are coupled via Ca2+ activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca2+-activated ACisoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca2+ over the entire physiological Ca2+ range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca2+ cycling proteins drive sarcoplasmic reticulum Ca2+ releases, which, in turn, activate ACs. This feed forward fail safe system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells. Recent studies indicate that basal phosphodiesterase (PDE) activity is increased in sinoatrial nodal cells (SANC) compared to ventricular myocytes (VM), and that the former express, Ca2+-activated adenylyl cyclases (AC), in addition to Ca-inhibited Type V AC. In SANC Ca2+ activates ACs and Ca-activated AC activity is present both in membrane microenvironments that are both rich in caveolin and in those with relatively low caveolin. Localized PDE activity can serve as a functional barrier to limit the diffusion of distal cAMP from the source of its generation by AC. The basal membrane microenvironment of AC activity may indicate that differing AC:PC activities occur within different membrane microenvironments and across the broad range of AC activity microenvironment, within SANC. Furthermore, with respect to G-protein-coupled receptor stimulation, beta-AR and ACs G proteins co-segregate with the high caveolin microdomain, i.e. within caveoli, the AC:PDE activity might be expected to be higher in caveolin-rich microenvironments than in areas in which caveolin is expressed to a lesser extent within both cell types. In order to analyze PDE and AC activities in SANC and VM in different intracellular microenvironment we collected separately lipid and water soluble fractions from cell lysates using sucrose gradient centrifugation method. Collected lipid microdomain fraction (rafts) compare to soluble protein fraction (soluble) had significantly lower protein concentration) and much higher concentrations of lipid raft markers caveolin and GM-1. We found that AC activity is much higher in rafts then in soluble fraction in both cell types and represent approximately 80-90% of the total activity. As in lysates, SANC demonstrated Ca2+-dependent AC activation but only in lipid-rich fraction. VM did not show significant Ca2+-dependent AC activity in both cases. It is important to mention that Ca2+ treatment did not change the pattern of AC activity distribution between fractions. But Ca2+-dependent AC activity (AC at 1 uM Ca2+ minus AC at 0 Ca2+) was significantly different between two cell types. PDE activity analysis demonstrated that the most of it in SANC was present in the Soluble fraction (approximately 80%). But the concentrations per mg of protein were not different between fractions in both cell types. Analysis of total AC and PDE activities (in combined fractions) demonstrated that in both cell types PDE activity is significantly higher then AC at all Ca2+ concentrations. And ratio of PDE to AC activities is significantly higher in soluble fraction then in rafts. The data demonstrate that even if PDE activity in total is higher then AC activity, subcellular environments are different in lipid rich and soluble domains in respect to the highly important intracellular second messenger cAMP. cAMP production is more favorably to occur in lipid rafts microdomain, but it is possible that its concentration is more tightly regulated in the Soluble microdomains. Comparison between these cells demonstrate that AC activity in all together combined fractions is approximately same in SANC and in VM, but obviously it is different is their Ca-dependent response. Analysis of PDE inhibitors influences on PDE IBMX-sensitive activity in SANC and VM revealed significant inhibitory effects of dipyridamole, rolipram, and combination of cilostamide with rolipram, demonstrating that PDE types 3, 4, and 5 to11 can be present in these cells.